Published online before print July 15, 2003
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Department of Oncological Sciences,
* Laboratory of Clinical Oncology and
Tumor Immunology, University of Torino Medical School, Institute for Cancer Research and Treatment, IRCC Candiolo, Italy
1Correspondence: Dept. of Oncological Sciences, Institute for Cancer Research and Treatment, Str. Provinciale 142, 10060 Candiolo, Torino, Italy. E-mail: francesco.leone{at}ircc.it
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Key Words: hematopoiesis • mature blood cells • CD34+ progenitors • proliferation • differentiation
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The expression of c-ErbB-2 in hematopoietic tissue has not been documented so far. Few studies raise the hypothesis that members of the EGFR family might be suitable targets of molecular biology techniques for cancer cell identification into blood samples [9 10 11 ]. We previously explored this hypothesis and discovered that the c-ErbB-2 transcript is systematically amplified on blood specimens by polymerase chain reaction (PCR)-based methods. We showed that low levels of c-ErbB-2 mRNA expression are detectable in bone marrow (BM), peripheral blood (PB), and mobilized PB (MPB) mononuclear cells (MNC) from healthy donors as well as from cancer patients [12 ]. This finding prompted us to investigate extensively the c-ErbB-2 expression and its functional state on blood cell subpopulations. Given the role described for c-ErbB-2 in embryogenesis [13 , 14 ], umbilical cord blood (CB) was also analyzed because of its different ontogenetic developmental status compared with adult blood.
We found that the c-ErbB-2 protein is present on the myeloid lineage of adult as well as umbilical blood. In quiescent CD34+ progenitor cells and in resting lymphocytes, which are c-ErbB-2-negative, we describe up-regulation of the receptor during the entry into cell cycle.
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Purification of CD34+ progenitors
CD34+ cell fraction was obtained from eight BM, 30 CB, and 13 MPB and was isolated with superparamagnetic microbead selection using a high-gradient magnetic field and miniMACS column (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s instructions. MPB samples were obtained from healthy donors for allogeneic transplantation. The purification was verified by flow-cytomerty counterstaining with a CD34 phycoerythrin (PE) HPCA-2 (Becton Dickinson Biosciences, BD PharMingen, San Diego, CA) antibody. In the cell fraction containing purified cells, the percentage of CD34+ cells ranged from 95% to 98%.
In vitro culture of hematopoietic cells
For hematopoietic progenitor in vitro expansion, purified CB CD34+ cells derived from 13 CB samples were pooled, and 1 x 106 cells/mL were cultured as previously reported [15
] in the presence of 50 ng/mL Flt3 ligand (FL; kindly provided by Stewart D. Lyman, Immunex Corp, Seattle, WA), 20 ng/mL thrombopoietin (TPO), 50 ng/mL stem cell factor (SCF), and 10 ng/mL interleukin-6 (IL-6). TPO, SCF, and IL-6 were generous gifts from Kirin Brewery (Tokyo, Japan). The cells were analyzed at 4 and 7 days of culture to monitor the expression of c-ErbB-2 on CD34+ cells. For in vitro differentiation of hematopoietic CD34+ progenitors, 1 x 106 CD34+ cells derived from eight CB samples were pooled and cultured in Iscove’s modified Dulbecco’s medium (Gibco-BRL, Life Technologies, Gaithersburg, MD), supplemented with 10% fetal calf serum (FCS; HyClone, Logan, UT) in the presence of 3 U/mL erythropoietin (EPO; Rhone Poulenc Rorer, Milano) for erythroid differentiation, 20 ng/mL granulocyte macrophage-colony stimulating factor (GM-CSF; Sandoz, Basel, Switzerland) for myelomonocytic differentiation, or 20 ng/mL TPO for megakaryocytic differentiation. The cells were analyzed at 4, 7, and 11 days of treatment for the presence of specific differentiation antigens and c-ErbB-2 by flow cytometry.
To induce proliferation of GM populations and lymphocytes, PB cells were cultured in the presence of 20 ng/mL G-CSF (Granulokine, Roche SpA, Monza, Italy) and 5 µg/mL phytohemagglutinin (PHA; Sigma-Aldrich, St. Louis, MO), respectively [16 , 17 ].
Cell lines
The human breast cancer cell line SKBR-3 was cultured in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen, San Giuliano Milanese, Italy) with 10% FCS. The immortalized human epithelial cell line MCF-10A was cultured in 1:1 DMEM/F-12 (Invitrogen) plus 5 µg/mL transferrin, 0.5 µg/mL hydrocortisone, 10 µg/mL insulin (Sigma-Aldrich), and 5 ng/mL EGF (PeproTech Inc., Rocky Hill, NJ), supplemented with 5% horse serum (Invitrogen).
RNA preparation and cDNA synthesis
Total RNA was extracted using the Trizol reagent (Invitrogen), according to the manufacturer’s instructions. RNA samples were digested with RNase-free DNase I (Roche Diagnostics, Italy), undertaken to a second round of Trizol extraction, and tested for Bax promoter amplification as described previously [12
]. Total RNA was used as a template for synthesis of oligo(dT)-primed double-stranded cDNA in 1x reverse transcription (RT) buffer (Invitrogen) supplemented with 0.01 M dithiothreitol (DTT), 200 units cloned Moloney murine leukemia virus RT (Invitrogen), 100 pmol oligo(dT) primers (Roche Diagnostics), 1 mM each dNTP (Invitrogen), and 200 units RNase inhibitor (Invitrogen) in a final volume of 25 µL and was incubated at 42°C for 45 min. To assess integrity of synthetized cDNA, 2 µL (8%) was subjected to a 50-µL PCR reaction containing 1x PCR buffer (Invitrogen) supplemented with 1.5 mM MgCl2, 200 µM dNTPs, 20 pmol glyceraldehyde 3-phosphate dehydrogenase (GAPDH) forward-amplification primer (5'-GAAGGTGAAGGTCGGAGTC-3') and 20 pmol GAPDH reverse-amplification primer (5'-GAAGATGGTGATGGGATTTC-3'), and 0.5 units Taq DNA polymerase (Invitrogen). Reactions were heated to 95°C for 2 min and then subjected to 40 cycles of 94°C for 15 s and 60°C for 1 min. All PCR products were analyzed on a 2% agarose gel with ethidium bromide staining.
Real-time quantitative PCR analysis (RQ-PCR)
Quantitation of c-ErbB-2 mRNA expression was obtained by RQ-PCR. The reaction was performed in a 50-µL master mix (Roche Diagnostics) with 15 pmol each primer. The c-ErbB-2 was amplified with primer 5'-TGCTGGAGGACGATGACATG-3' sense and 5'-CTGGACAGAAGAAGCCCTGC-3' antisense; human GAPDH gene was amplified as control with sense and antisense primer (see above). The reactions also contained 200 mM detection probes labeled with fluorescent dyes, the reporter dye [carboxyfluorescein (FAM)] and the quenching dye [carboxytetramethylrhodamine (TAMRA)]: FAM-5'-CCTGGTGGATGCTGAGGAGTATCTGGTACC-3'-TAMRA for c-ErbB-2 and FAM-5'-CAAGCTTCCCGTTCTCAGCC-3'-TAMRA for GAPDH (Perkin Elmer, Foster City, CA). All PCR reactions were performed in triplicate in a 96-well microtiter plate on a ABI-Prism 7700 sequence detector system (Perkin Elmer). The thermal cycling conditions were described previously [12
]. To express the relative amount of target in different samples, results were normalized to GAPDH mRNA and compared with the SKBR-3 cell line designed as the calibrator.
The ABI-Prism 7700 software provided an amplification curve constructed by relating the fluorescence signal intensity (
Rn) to the cycle number. Rn value corresponds to the variation in the reporter fluorescence intensity normalized to the fluorescence of an internal passive reference. Cycle threshold (Ct) was defined as the cycle number at which the fluorescence signal was greater than 10 standard deviations of the mean background noise collected from the third to the 15th cycle.
To minimize variability as a result of the differences in the RT efficiency or RNA integrity among samples, results were normalized to a housekeeping control gene, i.e., GAPDH. To calculate the relative expression of the target gene mRNA normalized to GAPDH, the target Ct was subtracted from GAPDH Ct (Ct). The amount of c-ErbB-2 mRNA (X) for each sample (q) normalized to GAPDH mRNA (R) and relative to the calibrator (cb) is given by 2–Ct, where Ct = CtX – CtR, difference in threshold cycles for target and reference, and Ct = Ctq – Ctcb. Derivation of the formula for the comparative Ct method is illustrated in the User Bulletin #2 of the ABI-Prism 7700 sequence detection system.
Western blot analysis
Five to 10 million cells were lysed with lysis buffer (50 mM Tris-HCl, pH 7.5, 250 mM NaCl, 2 mM EDTA, 50 mM NaF, 0.1 mM Na3VO4) plus 0.5% Nonidet-P40, 1 mM DTT, and protease inhibitors (Sigma-Aldrich) for 15 min at 4°C and were centrifuged at 20,000 g for 15 min. Protein concentration was measured using the Bio-Rad DC protein assay kit (Bio-Rad Laboratories, Hercules, CA) and 10–50 µg each lysate was resolved upon 7% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electrotransferred to 0.45 µm polyvinylidene difluoride (PVDF) membranes (Amersham Pharmacia Biotech, Piscataway, NJ). The membrane was then incubated for 60 min with 1 µg/mL rabbit polyclonal anti-ErbB-2, (neu c-18 sc-284, Santa Cruz Biotechnology, Santa Cruz, CA) and then with 1 µg/mL horseradish peroxidase (HRP)-conjugated secondary anti-rabbit antibody (Amersham Pharmacia Biotech). Filter was revealed with an enhanced chemiluminescence reagent (Amersham Pharmacia Biotech) and was exposed to autoradiography film. The filters were stripped and blotted with polyclonal goat anti-ß-actin anitbody (Santa Cruz Biotechnology) and were revealed as described above. To analyze tyrosine phosphorylation of c-ErbB-2, MNC were washed and incubated in serum-free DMEM with 5 ng/mL EGF at 37°C for the indicated time intervals. Each lysate (500 µg) was subjected to immunoprecipitation with 1 µg rabbit polyclonal anti-ErbB-2 and was electrophoresed on 7% SDS-PAGE, transferred to PVDF membrane, and immunoblotted with 1 µg/mL monoclonal antiphosphotyrosine antibody (Santa Cruz Biotechnology) and then with 1 µg/mL HRP-conjugated secondary anti-mouse antibody (Amersham Pharmacia Biotech). For immunoprecipitation and Western blot analysis of purified CD34+ cells, to reach the goal of 5 million cells, we pooled CD34+ cells derived from several samples (between three and six for each cell lysate).
Flow cytometry
For cytometric analysis, 1–5 x 105 cells were washed in 1x phosphate-buffered saline containing 0.1% bovine serum albumin (Sigma Aldrich) and 0.01% sodium azide incubated with human immunoglobulin G and labeled with fluorescein isothiocyanate (FITC)-conjugated CD antibodies. Then, cells were fixed with 0.4% paraformaldehyde, permeabilized with 0.1% saponin, and incubated with normal goat serum, stained with 10 µg/mL polyclonal anti-ErbB-2 antibody followed by PE-conjugated goat anti-rabbit isotype-specific antisera. The following antibodies were used: anti-CD14, CD15, glycophorin A CD41, CD2, and CD19 (all from Dako A/S, Denmark) and anti-CD34 phycocyanin-5 (PC5)-conjugated antibody (Immunotech, Coultar Co., Marseille, France). As control, we stained cells with an irrelevant antibody, followed by PE-conjugated goat anti-rabbit, isotype-specific antisera.
To describe the recruitment of quiescent cells into cell cycle, the cells were fixed, permeabilized, stained with FITC-conjugated antinuclear protein Ki67 [17 ] (clone B56, Becton Dickinson Biosciences, BD PharMingen) and anti-c-ErbB-2 antibodies, and subjected to fluorescein-activated cell sorter (FACS) analysis.
For each sample, 10,000 cells were analyzed on a FACSVantage SE (Becton Dickinson, San Jose, CA). Cell acquisition and analysis were performed using CellQuest software program (Becton Dickinson).
Trastuzumab treatment
To assess the effect of Trastuzumab treatment on peripheral monocytes, cells were plated at 1 x 106 cells/well in six-well microtiter plates and incubated in the presence or absence of 300 µg/mL Trastuzumab (Herceptin, Roche SpA) for 120 h. Each experiment was performed at least three times. The cells were washed and stained with polyclonal anti-ErbB-2 antibody for FACS analysis.
Trastuzumab was also added to in vitro culture of hematopoietic CD34+ progenitors. After 1 week, the cells were assessed for clonogenic capacity. Colony-forming cell (CFC) assays were performed as described previously [18 ].
Statistical analysis
All CFC assays have been performed in triplicate. Results were compared using the Mann-Whitney U nonparametric test. All P values are two-tailed with a significance of P< 0.05.
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The c-ErbB-2 transcript was found in all blood samples analyzed. Levels of expression in MNC from PB, BM, and CB and from isolated monocytes, lymphocytes, and granulocytes from PB were significantly lower if compared with those of the SKBR-3 cell line (Fig. 1 ); in addition, the CD34+ cell fraction showed the lowest level of c-ErbB-2 mRNA, and no significant difference of this transcript was observed among CD34+ cells from various sources (data not shown).
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Figure 1. Quantitation of c-ErbB-2 mRNA in hematopoietic cells by RQ-PCR. c-ErbB-2 mRNA expression of hematopoietic cells was compared with that of normal breast epithelial (MCF 10A) and breast cancer cell line (SKBR-3). The amount of c-ErbB-2 mRNA for each sample, normalized to GAPDH mRNA, is calculated by Eq. 2–Ct (see Materials and Methods for 2–Ct calculation), assuming that the expression of the calibrator (SKBR-3 in this experiment) is = 1. Histogram represents means and standard deviations of six independent experiments for each cell type.
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Figure 2. c-ErbB-2 protein expression in mature hematopoietic cells. Western blot analyses of total protein extracted from PB, CB, and BM MNC. The SKBR-3 cell line was used as control. Total proteins (50 µg and 10 µg) were used for blood samples and SKBR-3 cells, respectively.
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Figure 3. Immunophenotypical characterization of c-ErbB-2+ blood populations on normal PB, CB, and BM MNC. Cells were double-stained with the indicated anti-CD and anti-c-ErbB-2 antibodies followed by PE-conjugated goat anti-rabbit isotype-specific antisera. Plots show representative phenotypes of PB, CB, and BM MNC. As control, cells were stained with irrelevant rabbit polyclonal antibody, followed by PE-conjugated goat anti-rabbit isotype-specific antisera. GLYCO, Glycophorin A.
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Figure 4. Expression of c-ErbB-2 protein on CD34+ cells. (A) Representative FACS analysis of MPB-, BM-, and CB-derived CD34+ cells. Highly purified CD34+ cells (more than 98%) were characterized for c-ErbB-2 expression by single-color staining. (B) Western blot analysis on BM, MPB, and CB CD34+ cells. For BM and MPB samples, 500 µg total lysates from purified CD34+ cells were immunoprecipitated (IP) with anti-c-ErbB-2 antibody and immunoblotted (IB) with the same antibody; for CB samples, 50 µg total lysates from CD34+ cells were resolved by SDS-PAGE and immunoblotted with anti-c-ErbB-2 antibody. Each lane corresponds to pooled BM-, MPB-, and CB-derived CD34+ samples.
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Table 1. Relative c-ErbB-2 mRNA Quantification by RQ-PCR in Cytokine-Stimulated Hemopoietic Cells
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Figure 5. Protein expression of c-ErbB-2 in cultured hemopoietic cells. CD34+ cells were isolated from different CB samples, pooled, and cultured in the presence of FL, TPO, SCF, and IL-6. The cells were double-stained with PC5-conjugated anti-CD34 (horizontal axis) and anti-c-ErbB-2 (vertical axis), before (T0) and during culture for FACS analysis (A). Aliquots of the same cells before and after 7 days of culture were collected for protein extraction and Western blot analysis (B).
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Figure 6. Developmental expression of c-ErbB-2 during myelomonocytic, megakaryocytic, and erythroid differentiation. CD34+ cells were isolated from CB and cultured in the presence of the indicated cytokines. Cells were double-stained and analyzed by flow cytometry for the expression of lineage-specific CD and c-ErbB-2 at different days of cytokine stimulation. Cells cultured in the presence of GM-CSF for myelomonocytic differentiation were analyzed for CD14 and CD15 expression; cells cultured for megakaryocytic and erythroid differentiation were stimulated with TPO and EPO, respectively, and analyzed for CD41 and glycophorin A (GLYCO), respectively. T0, Analysis before cytokine stimulation.
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Figure 7. Ki67 nuclear antigen and c-ErbB-2 profile during the entry into cell cycle of cultured CD34+ cells. CD34+ cells, derived from CB and MPB, were cultured for the indicated days in the presence of FL, TPO, SCF, and IL-6. Cells were fixed, permeabilized, and double-stained with anti-c-ErbB-2 and anti-Ki67 for FACS analysis. The cell percentage of each quadrant is indicated. T0, Fresh CD34+ cells before cytokine stimulation.
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c-ErbB-2 activation analysis on blood cell
The c-ErbB-2 receptor tyrosine phosphorylation was examined by treating fresh MNC with EGF. Immunoprecipitation with anti-c-ErbB-2 and Western blot analysis on fresh MNC showed that p185 was phosphorylated and that p185 phosphorylation increased upon EGF exposure (Fig. 8
). Based on these data, we investigated whether EGF treatment had an effect on monocyte proliferation and adhesion or CD34+ progenitor clonogenic potential. Monocyte proliferation and adhesion to human endothelial cells as well as CFC clonogenic potential of CD34+ precursor cells were not significantly affected by EGF treatment compared with controls (data not shown).
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Figure 8. Tyrosine phosphorylation of c-ErB-2 receptor on PB MNC. Fresh MNC were exposed to 5 ng/ml EGF for the indicated time; total protein lysates were immunoprecipitated (IP) with anti-c-ErbB-2 antibody and immunoblotted (IB) with antiphosphotyrosine antibody ( -P-Tyr; upper) or anti-c-ErbB-2 antibody (lower). T0, Fresh MNC before EGF exposure.
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Figure 9. Down-regulation of c-ErbB-2 receptor on peripheral monocytes by Trastuzumab. Peripheral monocytes were incubated in medium supplemented with 10% FCS and with or without 300 µg/mL Trastuzumab (HERC) for 120 h. Cells were analyzed by flow cytometry for c-ErbB-2 levels.
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Table 2. Trastuzumab Effect on CFC Assays
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Here, we undertook the first systematic evaluation of the expression pattern of c-ErbB-2 in normal hematopoietic tissues. Quantitative PCR showed that MNC derived from PB, BM, and CB express low levels of c-ErbB-2 transcript as compared with epithelial cells. Hematopoietic CD34+ precursors from all blood sources express the lowest level of this transcript. By Western blot analysis and immunoprecipitation studies, the receptor was detected in most of blood samples, although with variable intensity of expression. By flow cytometric analysis, we observed coexpression of the myeloid markers and c-ErbB-2. The most stable and abundant expression was found in peripheral monocytes; by contrast, lymphocytes and their precursors did not coexpress c-ErbB-2.
To analyze c-ErbB-2 expression during hematopoiesis, we studied CD34+ precursors derived from BM, MPB, and CB samples. We found that c-ErbB-2 protein is expressed in only 2–5% adult CD34+ cells and in up to 40% CD34+ cells derived from CB. We demonstrated that the recruitment into cell cycle of quiescent cells is accompanied by the up-regulation of c-ErbB-2 and that this up-regulation is earlier in CB than in adult-derived hematopoietic precursors. Other studies demonstrated that a substantial fraction of the CD34+ cells derived from CB with a flow cytometric DNA content typical of the G0/G1phase is cycling [25 ]; so the higher c-ErbB-2 expression we found in CB seems to reflect its higher responsiveness to cytokine stimulation. The c-ErbB-2 up-regulation on hematopoietic precursors was confirmed during in vitro lineage-differentiation assays. These data suggest that c-ErbB-2 might participate into the hematopoietic cell proliferation mechanisms. We explored this hypothesis also in mature hematopoietic cells, and we found that c-ErbB-2 mRNA and protein expression increased upon mitogenic stimulation.
The results obtained from tyrosine kinase activation studies of c-ErbB-2 receptor on PB MNC clearly indicated the basal tyrosine phosphorylation status of c-ErbB-2 protein in a monocyte subpopulation. When these cells were exposed to EGF, increase in c-ErbB-2 tyrosine phosphorylation was observed. That EGF might act as a growth factor for hematopoietic cells is excluded in our experiments. In fact, the treatment of monocytes did not produce any effect on their proliferation and adhesion capacity. Similarly, EGF alone or in combination with hematopoietic growth factors did not influence the clonogenic or differentiation potential of precursor cells (data not shown). However, we cannot exclude a role for ligands of the EGFR family other than EGF in phosphorylation of the receptor and possibly in triggering some function of hematopoietic cells.
It is known that c-ErbB-2 is down-regulated following transactivation with EGF as a result of lysosomal targeting [26 ]. Down-regulation of the receptor is described to be impaired in c-ErbB-2-overexpressing cells, such as the SKBR-3 cell line [27 , 28 ]. This seems to depend on the inhibition of specific sorting steps in the endocytic pathway. In our system, although c-ErbB-2 was transactivated following EGF treatment, there was no measurable change in c-ErbB-2 expression. By contrast, Trastuzumab, a humanized anti-HER2 mAb, recognizes and down-regulates c-ErbB-2 expression on monocytes. For these reasons, we examined whether c-ErbB-2 receptor density on the cell surface of monocytes could be as high as in overexpressing conditions to justify the basal activation of the receptor as a result of autophosphorylation. With the mean fluorescent intensity study, we evaluated that the c-ErbB-2 receptor is at least 1 log lower represented as compared with breast cancer cells in which autophosphorylation phenomena are known (data not shown). Basal c-ErbB-2 phosphorylation on hemopoietic cells might be restricted to proliferating cells.
Experiments aimed at abrogating the c-ErbB-2 expression and investigating functional consequences were not successful, although a trend toward inhibition of colony formation from hematopoietic precursors in the presence of Trastuzumab was seen. This probably is a result, at least in part, of the incomplete suppression of the receptor by using the mAb in a nonoverexpressing system. Whether c-ErbB-2 activation and signaling is essential for hemopoietic cells or whether it is a part of a redundant system needs to be determined experimentally through genetic disruption studies.
Received February 12, 2003; revised April 29, 2003; accepted June 9, 2003.
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